This grant proposal primarily addresses broad Challenge area (06) Enabling Technologies. Specifically, the proposal meets two sub-areas within this main Challenge: 06-NS-105 Importing important technologies into neuroscience;06-NS-106 Validating new methods to study brain connectivity. The mammalian nervous system is enormously complex in terms of both the shear number of neurons (billions) and the diversity of neuronal subtypes (thousands). However, with a few rare exceptions, cell type- or region-specific "drivers" are largely unavailable in the mammalian model organism, the mouse, that allow us to dissect the precise connections and functions of the mammalian brain. Our proposal is aimed at solving this unmet challenge. We will take advantage of the recent elegant comparative genomics work in which a set of human enhancer elements were identified that are highly conserved across multiple vertebrate species. Importantly, these elements can drive reporter expression reproducibly in highly specific regions in the mouse nervous system. We will utilize this exciting knowledge obtained from genome sciences to generate region-specific Cre/CreER transgenic mouse lines driven by such conserved and ultraconserved human enhancers using a novel high throughput testis-electroporation method. Moreover, we have also developed an efficient split- Cre/CreER system. This innovative system allows us to use the enhancer elements in a combinatorial Venn Diagram manner to express the split Cre/CreER halves with different enhancers, and the active Cre/CreER is only reconstituted in the overlapping domains of two enhancers. In this way, we will develop Cre/CreER lines with extremely restricted expression pattern in the nervous system. With these much-needed Cre/CreER lines, neuroscientists will be able to map and manipulate mouse neural circuits with unprecedented ability and accuracy and to gain quantum leaps in our knowledge about the mammalian brain. Health Relevance This proposal is to capitalize on existing knowledge from genome sciences and novel transgenic technologies to catalyze major progress in basic neuroscience. We will generate multiple transgenic Cre/CreER mouse lines in which Cre/CreER or split Cre/CreER is expressed under the control of ultraconserved enhancer elements. In this way, Cre/CreER transgenic lines with highly restricted expression pattern in specific regions of the mouse brain will be developed. More complete understanding of the connectivity and function of the complex mouse brain networks will be critical for understanding the human brain and for answering many longstanding questions in basic and clinical neuroscience